The present invention relates to new methods of sequencing in which the information embodied by each base is effectively magnified, and methods which are particularly suitable for sequencing long nucleic acid molecules in which sequence information for portions of the sequence and details on the portions, positions within the sequence is combined, and kits for performing such methods.
Ever since Watson and Crick clarified the structure of the DNA molecule in 1953, genetic researchers have wanted to find fast and cheap ways of sequencing individual DNA molecules. Sanger/Barrell and Maxam/Gilbert developed two new methods for DNA sequencing between 1975 and 1977 which represented a major breakthrough in sequencing technology. All methods in extensive use today are based on the Sanger/Barrell method and developments in DNA sequencing in the last 23 years have more or less been modifications of this method.
In 1988, however, DNA sequencing technology acquired an entirely new focus. Led by the US, eighteen countries joined together in perhaps the largest individual project in the history of science, the sequencing of the entire human genome of 3xc3x97109 bp (the Human Genome Project, also called HGP), in addition to several other smaller genomes. As of today, the objective is to be finished during the year 2003. In spite of the fact that the project ties up large scientific resources and carries a large price tag, the gains of the project are considered sufficiently important to justify the cost.
An important part of the project is to develop new methods of DNA sequencing that are both reasonably priced and faster than current technology in principle, these can be divided into gel based (primarily new variants of the Sanger/Parrell method) and non gel-based techniques. The non gel-based techniques probably have a greater potential and mass spectrometry, flow cytometry, and the use of gene chips that hybridize small DNA molecules are some of the approaches that are being tested. Methods that are substantially better than current methods would result in a revolution not only for gene research but also for modern medicine since they would provide the opportunity for extensive patient gene testing and may play an important role in identification and development of drugs. The economic potential of such methods are naturally very great.
Using the currently known sequencing techniques, it has proved difficult to extend the length of the sequences that can be read for each sequencing reaction, and most methods used today are limited to about 7-800 base pairs per sequencing reaction. Nor is it possible to sequence more than one sequence per sequencing reaction with the methods widely used today.
To sequence many or long sequences, it is generally necessary to perform many parallel sequencing reactions (e.g. to sequence a diploid human genome of 6 billion base pairs, several million parallel sequencing reactions would be necessary). This is a considerable bottleneck because the total number of processes, the use of enzymes and reagents, the number of unique primers required, etc. are often directly proportional to the number of sequencing reactions that have to be performed. Furthermore, resources often have to be devoted to sequencing overlapping sequences. In addition, different types of organisation work must be performed, such as setting up and sorting a DNA library. It is also necessary to expend resources in order to isolate a possible target sequence if it is found among other sequences.
In order to illustrate the fundamental problems that limit the length of sequencing reactions, it is appropriate to divide the sequencing methods currently used and under development into two large groups (there are individual methods that fall outside this division, but they represent a small minority). In the first group, we have methods based on the size range of polynucleotides. The starting point is to make one or more polynucleotide ladders in which all molecules have one common and one arbitrary end. For example, the classical sequencing methods of Sanger and Maxam-Gilbert are based on four sequence ladders that represent each of the four bases A, C, G, and T.
The limiting factor with respect to the length of a sequencing reaction that can be read is that one must be able to distinguish between polynucleotides that only vary with one monomer. The longer the polynucleotides in the sequence ladder, the smaller the relative differences in size between the polynucleotides. Most of the methods for determining the size of molecules thus quickly reach a limit where it is not possible to distinguish between two adjacent polynucleotides.
In the other group the methods are based on a different principle. By identifying short pieces of sequences that are present in a target molecule, the target sequence can be reconstructed by utilising the overlaps between the sequence pieces.
Thus, in many sequencing methods target molecules are fragmented into smaller pieces, the composition of each fragment is deduced and by finding overlapping sequences the original sequence is constructed. For example, microarrays have been created with 65,536 addresses where each address contains unique octamers. All permutations with octamers (48=65,536) are thus covered. If the target molecules then are tagged with fluorescence and hybridised with the octamers, the information about what sequence pieces are present in the target sequence can be obtained by registering the addresses that have been labelled with fluorescence.
An important limiting factor with respect to the length of sequencing reactions that can be read is the following combinatorial problem. The longer the sequencing reaction that is to be performed, the longer the sequence pieces must be in order to make reconstruction of the target sequence possible. However, the number of permutations that have to be tested increases exponentially with the length of the sequence pieces that are to be identified. This increases the need for unique addresses on the microarrays in a corresponding manner.
An alternative use for microarrays is resequencing of known sequences, e.g. by screening for gene mutations in a population. For this purpose, oligonucleotides can be adjusted to the known sequence so that the number of addresses required can be reduced and the length of the sequence pieces that are identified can be increased. However, designing microarrays for specific purposes is expensive and resource demanding, and at present there are only microarrays for a few DNA sequences. Since the human genome consists of somewhere between 100-140,000 genes, it would be very resource demanding to mass-sequence human genomes in this manner.
Another disadvantage with using microarrays is that the limitations of current construction technology (e.g. photolithography) does not make it possible to create pixels of less than about 10xc3x9710 micrometers. Thus, only a fraction of the resolution potential of the fluorescence scanner is utilised. Current fluorescence scanners are capable of distinguishing pixels of 0.1xc3x970.1 micrometer, which means that microarrays can contain 10,000 times as much information as they currently contain.
It would therefore be advantageous to develop new methods/principles of identifying long sequence pieces where the combination problems mentioned above could be avoided. It would likewise be advantageous to develop new methods/principles that make it possible to sequence long target sequences without having the length of the sequence pieces that must be identified increase exponentially with the length of the target sequence.
Another sequencing method (for example as embodied in U.S. Pat. No. 5,714,330) that is based on identification of sequence pieces consists of distributing fragmented target DNA over a reading plate. Thereafter, the target DNA is treated so that a fluorescence signal representing one or several of the first base pairs is fixed to the target DNA. The fluorescence signals for each position is read before the procedure is repeated with the next base pair(s) in the target DNA. When the DNA molecules have fixed positions on the reading plate, it becomes possible to construct longer pieces with sequence information by running several cycles.
The ability to read several base pairs per cycle is limited because the number of unique fluorescence signals that are required increase exponentially with the number of base pairs. In order to read one base pair, four colours are required, two requires 16, three requires 64, etc. It is uncertain whether current technology makes it possible to distinguish between 64 different fluorescence colours. Regardless, the demands on reading time and the costs would increase considerably with the use of multiple colours. The solution would then be to perform many cycles. This in turn means an increase in the number of enzymatic steps and fluorescence readings.
Even if it were possible to identify relatively long pieces of sequence with the above-mentioned strategy, important problems would be encountered in the reconstruction. Biological DNA is very non-randomised in its composition. Short and long-sequences are often repeated in several places on a xe2x80x9cmacroxe2x80x9d and xe2x80x9cmicroscopicxe2x80x9d level. Reconstruction is particularly difficult in areas with repetitive DNA sequences. These can often be of biological interest; e.g. the length of trinucleotide xe2x80x9crepeatsxe2x80x9d.
A new approach which allows the above mentioned problems to be overcome has now been developed. Surprisingly it has been found that if sequence information is obtained which is linked to positional information (ie. information of the position of that sequence within a target sequence), long sequences can be identified accurately. Furthermore, the present invention provides new methods of sequencing which may be used with or without positional information in which the signal associated with one or more bases is amplified, referred to herein as magnification.
Thus according to a first aspect the present invention provides a method of sequencing all or part of a target nucleic acid molecule comprising at least the steps of:
a) determining the sequence of a portion of said nucleic acid molecule;
b) determining the position of said portion within said nucleic acid molecule; and
c) combining the information obtained in steps a) and b) to obtain the sequence of said molecule.
Conveniently, the sequence and position of multiple portions are determined and this information is combined.
As used herein the target nucleic acid molecule refers to any naturally occurring or synthetic polynucleotide molecule, e.g. DNA, such as genomic or cDNA, RNA, e.g. mRNA, PNA and their analogs, which where appropriate may be single, double or triple stranded. The part to be sequenced preferably comprises all of the target molecule, but may for example be less than the entire molecule, e.g. between 4 bases and 1 kb, e.g. 4 to 100 bases.
Preferably the portion which is sequenced has 4 or more bases and/or the position of said portion within said target molecule is determined with an accuracy of less than 1 kb (ie. to a resolution of less than 1 kb), particularly preferably less than 100 bases, especially preferably less than 10 bases. At a resolution of a few kb or better, it is usually not necessary to obtain sequence information on fragments of longer than 8-10 bases which is readily achieved by the methods described herein.
Sequence information may be obtained in any convenient manner and is appropriately obtained for one or more bases, especially preferably 2 or more bases, e.g. 2 to 20 bases, conveniently 4 to 10 bases. As will be appreciated it is imperative that the sequencing technique described above which relies on placement of the sequence portions within the target molecule, allows the retention of positional information which may be assessed simultaneously or separately to the sequence information. A number of appropriate techniques are described below.
Positional information may similarly be obtained in a number of convenient ways and these methods are also described below.
As mentioned above, in this aspect of the invention, the sequence which is obtained must be informationally linked to its position in the target sequence. This may be achieved in a number of ways, for example by sequencing the end or internal region of a nucleic acid molecule and establishing its position by reference to a positional indicator which may for example be the size of the molecule (e.g. length or volume), the intensity of a generated signal or the distance to a positional marker or anchor. Sequence information may be obtained by conducting one or more cycles of a sequencing reaction.
As mention previously, one of the difficulties in sequencing long molecules is that it becomes increasingly difficult to distinguish the different relative sizes of molecules which vary only by a single base as they become longer. In one embodiment, the present invention overcomes this particular problem by xe2x80x9cmagnifyingxe2x80x9d the difference in size, intensity, length or signal between molecules. Thus, in a preferred aspect the present invention provides a method of sequencing all or part of the sequence of a target nucleic acid molecule wherein 2 or more bases (e.g. 3 or more, preferably 4 or more) are sequenced per cycle of sequencing and/or the signal associated with each base is magnified.
As used herein a xe2x80x9ccyclexe2x80x9d of sequencing refers to execution of the series of steps resulting in an end product which may be processed to obtain sequence information, e.g. by generating or reading a signal therefrom. Preferably in magnification and sequencing reactions described herein more than one cycle is performed, e.g. 2 or more cycles, especially preferably more than 4 cycles, e g. up to 10 cycles.
xe2x80x9cMagnificationxe2x80x9d of a signal associated with a base refers to enhancement of a signal which is associated with, or may be attributed to, a single base. This may for example be an increase in size (where the signal is the size of the base) or development of a new signal, e.g. the addition or association of a label or other signalling means with that base.
Increasing the length of the sequence portions that are identified can compensate for low precision in size determination. Thereby, the potential of mass spectrometry, gel sorting and similar methods can be utilized, at the same time as enabling the use of methods of size determination that currently are not sufficiently precise, e.g. flow cytometry, DNA stretching, etc.
Magnification of the difference between molecules can be achieved in a number of ways. Firstly, several bases may be sequenced (e.g. 4 or more) per cycle such that the resultant molecules differ in length by 2 or more bases and can hence be discriminated e.g. in a sequencing ladder which simultaneously provides positional information (see for example Example 17). Alternatively the information embodied by each base may be magnified making discrimination easier. Examples of these different techniques are described below.
Sequencing of 2 (e.g. 4) or more bases per cycle can be performed by any convenient technique. In sequencing methods relying on positional information, any technique may be used providing the sequence information which is obtained can be related back to the position of those bases within the target molecule. In such cases, for example, hybridization to complementary probes (e.g. carried on a solid support) may be employed in which the identity of the probes to which the target molecules are bound are indicative of the terminal sequence of a target molecule. For example solid supports carrying probes complementary to all 2-base permutations, ie. carrying 16 different probes may be used. Similarly, probes to all 4-base permutations, ie. 256 different probes could be attached to a solid support for capture of target molecules with complementary sequences.
In an exemplary procedure, all target molecules that do not end with AAAA (SEQ ID NO:1) (when the probe ends in TTTT (SEQ ID NO:2)) would not bind and would be removed. Similarly at other addresses, target molecules having particular end sequences would bind selectively. The target molecules may be double stranded (with single stranded overhangs) or single stranded such that sequences could be bound to and identified at the terminal ends or also internally, respectively. If PNA was used as the complementary probe, since such molecules are able to bind to double stranded forms, internal sequences, of double stranded forms could also be bound. In general terms this technique is referred to herein as sorting based on one or cycles. This technique may be coupled to other techniques as described herein.
As a reverse of the above described technique, sequencing may be performed by fixing single stranded target DNA to a solid substrate. The target DNA may then be mixed and hybridized, for example with 16-fragment adapters. Adapters are described hereinafter, but generally refer to molecules which adapt the target sequence to a signal-enhanced or magnified target sequence. Adapters that have not been hybridized are then washed away from the solution. This leaves only adapters with overhangs that are complementary to the single strand DNA. With the aid of for example the analysis methods described herein one can establish which adapters remain in the solution and consequently what sequence pieces of 16 base pairs are contained in the DNA.
This sequencing technique represents a preferred feature of the invention when performed in conjunction with positional information or when used as a sequencing technique alone. In this method the information carried by a single base is magnified, e.g. by multiplication of that base or replacement or enhancement of that base with a magnifying tag which can be used to generate a signal. (Magnification as referred to herein is also in some instances referred to as xe2x80x9cconversionxe2x80x9d.)
Magnification of a target nucleic acid molecule may be achieved by multiplying, e.g. doubling a target molecule, or portion thereof containing the portion to be sequenced, one or more times. It will be appreciated that detecting the differences between molecules of e.g. 10 and 11 bases is more-difficult that detecting the difference between magnified molecules of 320 and 352 bases (doubled 5 times). The principled doubling can therefore be used, e.g. to improve most DNA analysis methods. One appropriate technique for achieving this is described in Example 11. Other appropriate techniques may also be used.
This method can therefore be used to improve most methods based on detecting size differences between nucleic acid molecules, e.g. gel or non-gel based techniques. This strategy also makes it possible to analyse nucleic acid material using techniques that are not sensitive enough to distinguish the difference of a few base pairs. For example, an improved Maxam-Gilbert method is possible in which a single stranded nucleic acid molecule (e.g. with 5xe2x80x2-biotin) is attached to a streptavidin bearing plate. Sequencing is then conducted and the plates washed after which the resultant nucleic acid molecules are doubled, e.g. 10 times, resulting in steps of 1024 base pairs. These lengths may be determined by one of the analysis techniques described below.
Thus viewed from a further aspect the present invention provides a method of sequencing all or part of a target molecule as described herein wherein the signal associated with each base (or more than one base) is magnified by increasing the number of times that said base appears in said sequence.
As used herein the xe2x80x9csignalxe2x80x9d attributed to a particular base (or more than one base) refers to the possibility of detecting that base (or collection of bases) by virtue of its properties either directly or indirectly. Thus this could refer to its properties of for example size, charge or spatial configuration which might be detected directly or indirectly or by association of one or more further molecules, e.g. labelling moieties with said base from which a signal may be generated directly or indirectly. Thus a signal may be provided which may be detected directly or a signalling means may be provided through which a signal may be generated. The signal may be unique to more than one base, ie. the signal may be indicative or representative of a pair of bases, e.g. a signal for AA may be used which is different to the signal used for AT etc. Different mechanisms for associating such molecules and signals that may be generated are described in more detail below.
A further preferred magnification technique involves the association of one or more unique signals (or means for producing such signals) with one or more bases in a sequence. When said signals are associated with more than one base, this may be achieved by using a series of signals (or signalling means) each corresponding to one or more bases or a single signal (or signalling means) unique to two or more bases. Conveniently these signals are carried on magnifying tags which may become attached to the sequence through an adapter molecule. xe2x80x9cAssociationxe2x80x9d as used herein refers to both replacement of said base (or more than one base) with said signal (or signalling means) or addition of said signal (or signalling means) to said base (or more than one base) such that they coexist. The signal (or signalling means) need not necessarily be attached directly to (or specifically replace) the base (or more than one base) with which it is associated and association may be indirect, e.g. through the intermediacy of one or more further molecules. Association may be through any appropriate chemical interactions, e.g. hydrophobic, ionic, covalent etc., but preferably is by covalent interaction with the target nucleic acid molecule or associated molecule.
xe2x80x9cCorrespondingxe2x80x9d as used herein refers to the relationship between a base and a signal, for example as provided by a magnifying tag, which may be read as indicative of the presence of that particular base. Alternatively in the-context of the mapping procedure this refers to the relationship between a nuclease and a signal used as a marker indicative of cleavage with that nuclease.
As used herein a xe2x80x9cmagnifying tagxe2x80x9d is a single molecule or complex of molecules which comprise a tag portion which provides a means for generating one or more signals, e.g. carrying a label or a site to which a label may be bound. Means for generating one or more signals may be incorporated in instances in which information other than sequence information is required, e.g. as an indicator of information relating to the target molecule or the cleavage protocol which is used. A magnifying tag may inherently carry a further portion for specifically associating with one or more nucleotide bases, e.g. when the magnifying tag is a polynucleotide. In this case the tag is considered to additionally comprise the adapter as described herein. Alternatively, the magnifying tag may be attached to, or contain means for attachment to, an adapter which allows binding to a target sequence.
In general terms an example of the process may be described as follows. Base pairs in the target nucleic acid material are associated with four different tags (hereafter called magnifying tags) that represent each of the four bases Adenine, Cytosine, Guanine, and Thymine. Thus, where there was an A-T base pair xe2x80x9cmagnifying tag Axe2x80x9d is associated, C-G is associated with xe2x80x9cmagnifying tag Cxe2x80x9d, etc. Thereby new DNA molecules are generated where the original base order of e.g. ACGTT (SEQ ID NO:3) is augmented by xe2x80x9cmagnifying tag Axe2x80x9dxe2x80x94xe2x80x9cmagnifying tag Cxe2x80x9dxe2x80x94xe2x80x9cmagnifying tag Gxe2x80x9d, etc. Each magnifying tag provides a means of producing a signal and may in a preferred feature be a polynucleotide molecule. In that case the length of the four tags may vary from two base pairs to several hundred kbp (or more if desired), according to requirements. Correspondingly, the DNA fragments can contain reporter genes and other biological information or consist only of sequences without a known biological function.
Any convenient magnifying tag may be used, but it is of course imperative for sequencing purposes that at least 4 unique tags exist, ie. for each base. Of course the tag to be used depends on the sequencing technique and, where this is performed, the method used to extract the positional information.
Tags may be provided in a number of alternative forms. The tag has means for direct or indirect detection through the generation of unique signals, ie. the tag comprises one or more signalling means. Fluorescence, radiation, magnetism, paramagnetism, electric charge, size, and volume are examples of properties with which the magnifying tag particles can be equipped in order to be able to detect them and separate them from each other. These properties may be present on one or more labels present on the magnifying tags, the signals from which may be detected directly or indirectly. Appropriate labels are those which directly or indirectly allow detection and/or determination of the magnifying tag by the generation of a signal. Such labels include for example radiolabels, chemical labels (e.g. EtBr, TOTO, YOYO and other dyes), chromophores or fluorophores (e.g. dyes such as fluorescein and rhodamine), or reagents of high electron density such as ferritin, haemocyanin or colloidal gold. Alternatively, the label may be an enzyme, for example peroxidase or alkaline phosphatase, wherein the presence of the enzyme is visualized by its interaction with a suitable entity, for example a substrate. The label may also form part of a signalling pair wherein the other member of the pair may be introduced into close proximity, for example, a fluorescent compound and a quench fluorescent substrate may be used.
A label may also be provided on a different entity, such as an antibody, which recognizes at least a region of the magnifying tag, e.g. a peptide moiety of the magnifying tag. If the magnifying tag is a polynucleotide, one way in which a label may be introduced for example is to bind a suitable binding partner carrying a label, e.g. fluorescent labelled probes or DNA-binding proteins. Thus, alternatively the tag may carry a molecule or itself be a molecule to which a label may be attached, e.g. by virtue of its sequence. Labels may be attached as single molecules or in the form of microparticles, nanoparticles, liposomes or other appropriate form of carrier.
In a preferred aspect, the magnifying tags are themselves nucleic acid sequences of at least 2 bases, e.g. from 30 to 1000 bases, preferably 6 to 100 bases, especially preferably 10 to 30 bases in length. These sequences may have one or more labels attached to them, e.g. by the use of fluorescing probes, proteins and the like complementary to that sequences, from which one or more signals may be generated. Alternatively, protein molecules may comprise the tag or be attached to the tag and may be recognized, e.g. by immunoreagents or by another appropriate binding partner, e.g. DNA: DNA-binding proteins. Other properties of such tag molecules may also be examined, e.g. cleavage patterns (by restriction enzymes, or proteinases), charge, size, shape etc.
The magnifying tags may also contain information by virtue of its sequence which can be used to generate a signal. Thus, another alternative strategy is to create chains that contain reporter genes, cis-regulatory elements, and the like. These can then be transfected/transformed into cells where the composition of e.g. reporter genes or cis-regulatory elements are converted into one or several signals. Whilst this technique requires a transformation/transfection step, the cells may be programmed to perform the complete sequencing reaction including the conversion step (ie. the addition of magnifying tags). A huge repertoire of signals may be generated, such as the use of genes expressing fluorescence proteins or membrane-proteins that can be labelled with fluorescence, genes expressing antibiotic resistance etc. Signal quality, quantity and position can be exploited in addition to changes with time and other properties to indicate the presence of particular bases in a sequence.
Conveniently solitary cells are used in these methods, although multicellular organisms or structures could also be used. Non-living cell equivalents could also be used for the generation of the labels or signals, e.g. using nanotechnology. Where appropriate, signals which are generated may be directed to different locations for identification, e.g. by the use of different promoters. Examples of how this technique might be performed is shown in Example 18.
Whilst conveniently 4 magnifying tags specific to each of the nucleotide bases may be used, as mentioned above, where appropriate magnifying tags which may be used to generate signals unique to more than one base may be used. Thus for example, for reading methods where e.g. 16 different fluorophores can be used, it may be appropriate to use 16 different tags which are used to generate 16 different signals that represent all permutations of two base pairs.
In other contexts, it may be appropriate to use fewer than four different tags. For example, only two magnifying tags where one is for A/T while the other is for C/G. Another alternative is to use less than 4 unique signal events to create 4 magnifying tags which give rise to 4 unique signals (in instances in which individual bases are tagged) by virtue of particular combinations of those signal events. For example, sequencing information may be converted into a binary system. In this system, adenine may-be converted to a series of signal events xe2x80x9c0xe2x80x9d+xe2x80x9c0xe2x80x9d, cytosine to xe2x80x9c0xe2x80x9d+xe2x80x9c1xe2x80x9d, guanine to xe2x80x9c1xe2x80x9d+xe2x80x9c0xe2x80x9d, and thymine to xe2x80x9c1xe2x80x9d+xe2x80x9c1xe2x80x9d. In principle, it is then enough to have one or perhaps two colours or unique signals to read the sequencing information. This may in turn mean that less costly fluorescence scanners can be used at the same time as reading is faster than if several signals had been used. The use of a single signalling means spatially arranged to provide at least 4 unique magnifying tags, e.g. to produce a binary type readout forms a preferred aspect of the invention, ie. said signal comprises a pattern made up of a single signal event which creates a unique signal on said magnifying tag. In this case a signal event refers to a measurable signal e.g. the fluorescence from a single molecule or other such label. When multiple magnifying tags, preferably 20 to 100 tags, are used these are preferably associated linearly, e.g. as a long DNA fragment, to allow-positional information to be preserved when this is required.
Association of the tag (albeit not necessarily directly, e.g. this may occur through an adapter) with the base (or more than one base) which it represents relies on specific base recognition via for example base-base complementarity. However, complementarity as referred to herein includes pairing of nucleotides in Watson-Crick base-pairing in addition to pairing of nucleoside analogs, e.g. deoxyinosine which are capable of specific hybridization to the base in the target nucleic acid molecule and other analogs which result in such specific hybridization, e.g. PNA, RNA, DNA and their analogs.
Thus probes could be used which are for example made up of DNA, RNA or PNA sequences, or hybrids thereof, such as oligonucleotides of for example 4 to 20 bases, preferably 6 to 12 bases in length which bind to specific regions of a target molecule (where the complementary sequence is present) and have attached thereto a magnifying tag, or series of tags, in which each tag represents one or more of the nucleotide bases to which the probe binds. In this case the probe acts as an adapter molecule facilitating binding of the tag to the target sequence. Alternatively, mixes of degenerate probes may be used with only one or more specific invariant bases at a particular position, e.g. NNNNAA. Preferably the number of magnifying tags which are present correspond to the number of specific bases of the probe to which they are attached, which become bound. However, if unique tags are made for 2 or more base permutations correspondingly less tags are required.
This technique may be used to identify discrete portions or parts of a target molecule or to obtain the sequence of all or essentially all of the target molecule""s sequence.
A more elegant sequencing alternative however is provided by the production of a contiguous magnifying tag chain the signals from which can be read to derive the sequence. Although there are other ways of achieving this effect the most convenient technique involves the insertion of the magnifying tags into the target molecule. Especially preferably this reaction is performed cyclically allowing the conversion and later sequence reading of a series of bases.
In order to insert the magnifying tags into the target molecule in association with the base (or more than one base) to be magnified it is necessary to use complementarity to, or recognition of, that base (or more than one base) and surrounding bases. This complementarity may be used to directly introduce a magnifying tag or may be used to initiate a procedure which ultimately introduces a tag corresponding to that base (see Example 4).
Conveniently this is achieved by creating an overhang (ie. a region that is single stranded) in the target nucleic acid molecule that could be ligated to a magnifying tag. (Such an overhang is however not necessary where a tag molecule or its intermediary, e.g. its adapter, can recognize and bind to double stranded forms, e.g. PNA). One method is to ligate the ends of the target molecule with short DNA molecules that contain a binding site for a restriction enzyme that cleaves outside its own recognition sequence, e.g. class IP or IIS restriction enzymes. These enzymes exhibit no specificity to the sequence that is cut and they can therefore generate overhangs with all types of base compositions. The binding site can be located so that an overhang is formed inside the actual target molecule, e.g. DNA when the DNA molecules are incubated with the restriction enzyme in question. In practice, it is probably preferable to choose enzymes that generate 3-4 base pair overhangs (see Example 19 which shows the general procedure for producing such overhangs on target molecules which have been amplified and attached to a solid support).
Over 70 classes of IIS restriction endonucleases have been identified and there are large variations both with respect to substrate specificity and cleaving pattern. In addition, these enzymes have proved to be well suited to xe2x80x9cmodule swappingxe2x80x9d experiments so that one can create new enzymes for particular requirements (Huang-B, et al.; J-Protein-Chem. 1996, 15(5):481-9, Bickle, T. A.; 1993 in Nucleases (2nd edn), Kim-YG et al.;PNAS 1994, 91:883-887). Very many combinations and variants of these enzymes can therefore be used according to the principles described herein.
Class IIS restriction endonucleases have been used for several different purposes. For example, as universal restriction endonucleases that can cleave a single strand substrate at almost any predetermined site (Podhajska, A. J., Szybalski, W.; Gene 1985, 40:175-182, Podhajska, A. J., Kim, S. C., Szybalski.,W.; Methods in Enzymology; 1992, 216:303-309, Szybalski, W.; Gene 1985, 40:169-173)
In sequencing contexts they have been used for the previously mentioned method described in U.S. Pat. No. 5,714,330. In these cases however the introduction of multiple magnifying tags which remained associated with the target molecule was not considered.
Cleavage with IIS enzymes result in overhangs of various lengths, e.g. from xe2x88x925 to +6 bases in length. Once an overhang has been created, magnifying tags, which may be carried on adapters, corresponding to one or more of the bases in the overhang may be attached to the overhang.
A number of different ways in which the magnifying tags may be incorporated using the IIS system or similar systems are described below.
The first described technique involves the use of adapters which carry one or more magnifying tags and which have a complementary overhang to the target nucleic acid molecule which has been modified to generate a single stranded region, ie. an overhang. The adapter itself also carries the recognition site for a further IIS enzyme which may be the same or different to the enzyme used to generate the overhang. An example of this technique is illustrated in Example 1.
Briefly, the target sequence is ligated into a vector which itself carries a IIS site close to the point of insert or the target sequence is engineered to contain such a site. The appropriate IIS enzyme is then used to cleave the IIS site which when appropriately placed results in an overhang in the target sequence. In one embodiment, at least one end of the cut vector is made blunt, e.g. by the use of a further restriction enzyme site adjacent to the IIS.
Appropriate adapters may then be used to bind to, and thereby allow magnification of, one or more bases of the overhang. In the case of single base magnification, degenerate adapters having single stranded portions of the form, e.g. for a four base overhang, ANNN (SEQ ID NO:4), TNNN (SEQ ID NO:5), CNNN (SEQ ID NO:6) and GNNN (SEQ ID NO:7) and magnifying tags A, T, C and G, respectively may be used. Alternatively the adapters may carry more than one magnifying tags corresponding to more than one of the overhang bases, e.g. having an overhang of ATGC (SEQ ID NO:17), with corresponding magnifying tags to one or more of those bases attached in linear fashion where appropriate.
Once the overhang of the adapter and the cleaved vector have been hybridized, these molecules may be ligated. This will only be achieved where full complementary along the full extent of the overhang is achieved and aids the specificity of the reaction. Blunt end ligation may then be effected to join the other end of the adapter to the vector. By appropriate placement of a further IIS site (or other appropriate restriction enzyme site), which may be the same or different to the previously used enzyme, cleavage may be effected such that an overhang is created in the target sequence downstream of the sequence to which the first adapter was directed. In this way adjacent or overlapping sequences may be consecutively converted into sequences carrying magnifying tags, the signals from which can subsequently be read to determine the sequence by the methods described later herein. The sequencing of overlapping sequences effectively allows proof-reading of sequences which have been read in previous cycles allowing verification.
A slight modification of this technique is shown in Example 2 in which a blunt end is not produced, but instead once the vector has been produced and cleavage effected with the IIS or similar enzyme, a further restriction enzyme is used which creates an overhang which is universally complementary to the terminal of all adapters which are inserted into the vector. This similarly allows ligation of the adapter and therefore magnifying tags into the vector.
A similar but more elaborate example is illustrated in Example 3. In this case non-complementary overhangs are created corresponding to adjacent stretches of DNA. These are both hybridized to adapters which have attached appropriate magnifying tags. Only one of the adapters contains the restriction enzyme site for the next cycle so that sequencing occurs unidirectionally. Clearly to allow the binding of adapters with these different properties, the overhangs of adjacent stretches of DNA must be discriminatable, e.g. be of different length. This can be achieved by using different restriction enzymes which result in different overhang lengths. The ends of the two different types of adapters are intentionally complementary and thus would hybridize and may be ligated to form the vector. The restriction site in the adapter which contains it is appropriately placed such that the cleavage site is displaced further into the target sequence to allow sequencing of adjacent sites.
Thus for example if overhangs of 5+4 are created, and the cleavage site is displaced 4 bases into target sequence, when the next 9 bases are converted to overhang and thereafter associated with magnifying tags, 5 of these bases will have been associated with magnifying tags in the previous cycle. This allows verification of the identity of the previous 5 bases when reading the sequence and thus introduces a proof-reading mechanism.
Other techniques using the IIS system include the use of Klenow fragment of DNA polymerase and relies on the fact that most DNA ligases are unable to ligate overhang of different sizes. This is shown for example in Example 5. In this technique an overhang is created which is longer than the overhang of the adapter. The target overhang is reduced by Klenow in the presence of one type of nucleotide. Only the target which has been appropriately extended by one base will bind to the adapter allowing identification of the base that was introduced by virtue of the corresponding magnifying tag attached to that adapter.
Other techniques illustrated in Examples 4-7 involve the hybridization of adapters carrying magnifying tags to a single stranded target which are then ligated to that target. The adapter then is used as a primer for a polymerase extension reaction to form double stranded molecules. A further alternative uses sorting adapters (which in this case need not necessarily be associated with magnifying tags and may simply be used for sorting) in which the adapters are attached to a solid support which have an overhang in excess of the overhang created on target molecules. Thus for example the adapters may have an overhang of 8-10 bases. If for example the DNA pieces (in double stranded form) have a 4-base overhang these molecules will only ligate if the bases complement one another at the innermost bases of the overhang. Polymerase extension is then performed. The prerequisite for a successful polymerase extension reaction is that the rest of the adaptor""s overhang is complementary to the DNA piece so that it can function as a primer. In this way polymerase extension will only occur if the target molecule""s terminal sequence is complementary to the adapter""s overhang.
Alternatively hybridization alone may be used and magnifying tags which are associated with stretches of the sequence which are adjacent may conveniently be ligated together.
A further alternative relies on the specificity of metabolic enzymes for their recognition sites. Such a technique is illustrated in Example using restriction enzymes. A number of alternative enzymes may however also be used such as transposases etc. In this method the target molecules that are to be sequenced are cleaved to produce blunt ends with four different standard restriction enzymes and ligated into 4 different DNA molecules each ending with a portion of the restriction site for one of 4 different restriction enzymes (which produce an overhang on cleavage). These are then ligated onto the target molecules. Where the target molecules end with bases which provide the remaining bases of the restriction site, a restriction recognition site will be produced. This can be determined by cleavage with that restriction enzyme. Only those molecules that have that recognition site in complete form will be cleaved. To recognize those molecules which have been cleaved, adapters may be used which are complementary to the overhang. These adapters may then carry one or more appropriate magnifying tags depending on the number of bases provided by the target molecule to complete the restriction site. The molecule may then be circularized to allow repeat cycling. Conveniently the adapters have within their sequence appropriately sited restriction sites for both blunt end and overhang producing restriction enzymes, such that reiterative cycles may be performed by allowing the introduction of magnifying tags corresponding to adjacent or overlapping target sequence regions.
The present invention thus relates in one aspect to a method of identifying a portion of a target nucleic acid molecule wherein an adapter molecule comprising a moiety which recognizes and binds to said portion and a moiety comprising one or more magnifying tags, preferably a chain of said tags representing the bases in said portion, is attached to, or substituted for, said portion.
Thus viewed from a preferred aspect the present invention provides a method of magnifying all or part of the sequence of a target nucleic acid molecule wherein one or more magnifying tags are associated with one or more bases in the target sequence, wherein said tags correspond to one or more bases in said target sequence. Preferably said magnifying tags together correspond to at least two, preferably at least 4 bases. Preferably said magnifying tags each correspond to at least two, preferably at least 4 bases. In an alternative embodiment each magnifying tag corresponds to one base and a chain of magnifying tags together corresponding to at least 4 bases, e.g. 8 to 20 bases is employed. This may for example be achieved by performing multiple cycles adding a single magnifying tag in each cycle or by using chains of tags which are associated in a single cycle.
Preferably said method comprises at least the steps of:
a) converting at least a portion of said target sequence to a form suitable for binding an adapter molecule, preferably to single stranded form;
b) binding to at least a portion of said region suitable for binding an adapter molecule, preferably said single stranded region, created in step a) an adapter molecule comprising one or more magnifying tags, or comprising a means for attaching one or more magnifying tags, which tags correspond to one or more bases of said target sequence, preferably corresponding to one or more bases of said region suitable for binding said adapter molecule, preferably said single stranded region, to which said adapter molecule binds or in proximity to said region;
c) optionally ligating said target molecule to said adapter molecule such that at least said magnifying tags remain associated with said target molecule;
d) optionally repeating step a), wherein said region suitable for binding said adapter, preferably said single stranded region, which is created includes one or more bases not associated with a magnifying tag according to step b);
e) optionally repeating steps b) to d) wherein said adapter molecule binds to an adjacent or overlapping region of said target molecule relative to the region to which the adapter molecule of the previous cycle bound.
Step e) may be omitted in some techniques, e.g where sequencing is achieved by coupling magnification and sorting, such that only one cycle of magnification is performed.
xe2x80x9cConversionxe2x80x9d to a form suitable for binding an adapter molecule is necessary only if a target molecule is not already in an appropriate form. Thus to bind PNA molecules, conversion of double stranded target molecules is not necessary. Similarly, if a molecule is single stranded conversion is not necessary to bind adapters which are oligonucleotides. In some cases however conversion may be required, e.g. by melting DNA fragments, to allow specific and selective binding of the adapter. It is not necessary to convert the entire molecule to a different form and in appropriate cases only a portion will be converted. This portion should comprise at least the length of the binding portion of the adapter, thus preferably 4 to 500 bases, e.g 6 to 30 bases in length. Reference in this context to conversion from one form to another should not be confused with use of the word conversion when used in relation to magnification.
As used herein an xe2x80x9cadapter moleculexe2x80x9d is a molecule which adapts the target sequence to a signal-enhanced or magnified target sequence. Adapter molecules as used herein are single molecules or complexes of molecules which may be the same or different in type. The adapter sequence comprises a binding moiety which binds to said target sequence, e.g. a protein recognizing a particular base sequence or more preferably a polynucleotide sequence complementary to one or more bases of the target sequence. Preferably the binding sequence is 3 to 30 bases, preferably 4 to 10 bases in length. Adapter molecules additionally comprise one or more magnifying tags or means for attaching such tags, e.g. sequences which are complementary binding partners. Preferably adapters contain one or nuclease recognition sites, especially preferably a restriction site (or at least recognition site) for a nuclease which cleaves outside its recognition site, especially preferably restriction sites of IIS enzyme or their analogs, particularly FokI and other enzymes described herein. Preferably sites for other restriction enzymes are excluded from the adapters.
Conveniently adapter molecules may be exclusively comprised of a nucleic acid molecule in which the various properties of the adapter are provided by the different regions of the adapter. However, as mentioned previously magnifying tags may take a variety of forms, which include labels such as proteins, etc. The adapter may thus provide the molecule to which magnifying tags may be bound, e.g. provide appropriate binding partners in addition to the region for binding to the target.
In step c) it is indicated that xe2x80x9cat leastxe2x80x9d said magnifying tags remain associated. It is thus envisaged that the adapter or portions thereof may be removed.
As used herein a xe2x80x9cchainxe2x80x9d of magnifying tags refers to tags which have been linked either before a cycle of magnification and attached to one adapter or linked together at the end of each cycle or a combination of both. The linkage may be by any appropriate means however attachment by covalent means is preferred.
Preferably the above method is used in sequencing methods of the invention which comprise the above steps in addition to determining the sequence of said target molecule by identifying the signals generated from the magnifying tags attached to said target sequence. In order to identify the magnifying tags a readable signal must be generated from the magnifying tags. This may be present inherently, for example where the tags carry a label with certain properties (e.g. a radioactive label), or may require further steps for its generation, e.g. the addition of further molecules (e.g. binding, partners themselves carrying labels) or processing of the magnifying tags into a readable form (e.g. conversion to a readable signal such as by expression of a reporter gene in which the signal which is read is the expressed protein).
Thus in a preferred aspect the present invention provides a method of sequencing all or a part of a target nucleic acid molecule in which at least a portion of the sequence of said target nucleic acid molecule is magnified, preferably by the use of one or more magnifying tags associated with one or more bases in the target sequence, wherein said magnified sequence is optionally converted into a readable signal and said sequence is determined by assessment of the signals which are generated.
xe2x80x9cAssessingxe2x80x9d as used herein refers to both quantitative and qualitative assessment which may be determined in absolute or relative terms.
Ligation may be achieved chemically or by use of appropriate naturally occurring ligases or variants thereof. Whilst ligation represents only a preferred feature of the invention, this is conveniently used to increase specificity. Compared to hybridization, specificity is increased by a factor of ten if ligation is based on T4 DNA ligase. This is important since the sequencing methods that are based on hybridization in many cases are associated with an unacceptably high error rate. Furthermore, by using thermostable ligases, such as Pfu, Taq, and TTH DNA ligase specificity will be improved while efficiency increases dramatically so that the incubation time is reduced.
This method of sequencing using magnifying tags offers a number of advantages over known methods of sequencing. More than one base may be converted or magnified in each cycle thus reducing the number of cycles necessary for sequencing a particular length of target molecule. Depending of the choice of magnifying tags and the signals they produce, simplified read-out signals may be produced, e.g. the signals may be in the form of a binary read-out, ie. unique signals are generated for one or more bases by appropriate combination, e.g. linear or positional layout, of a single signalling event, e.g. fluorescence. This reduces the number of unique signalling events which are required. Thus instead of needing for example 16 different labels for each 2 base combination, or 64 different labels for each 3 base combination, in the present invention 16 or 64 or more unique signals may be generated by providing on each magnifying tag a pattern of a means for producing a single signalling event, e.g. a pattern of sites for binding a fluorescent probe.
The signal information may be tightly packed. The tags are not limited to only labelled nucleotides allowing greater flexibility in the types of magnifying tags which may be used and signals which may be generated. In some embodiments, even when cycling is not performed, large portions of a sequence may be sequenced by using chains of magnifying tags to those portions thus avoiding the complex reactions involved in repeat cycling and also the need to relate the information from each cycle to a particular target sequence, e.g. using target molecules fixed to a reading plate, which limits how the signal may be read (e.g. micro/nanopores or flow cytometers could not be used)
In preferred aspects of the invention, conversion of the target molecule to at least a partially single stranded form is achieved by using a single stranded molecule or by creating an overhang, e.g. by using an appropriate nuclease with a cleavage site separate from its recognition site, such as IIS enzymes.
Preferably when the reaction is performed cyclically, the magnifying tags of each cycle are joined, e.g. by association or ligation, together, e.g by the production of a single chain containing them. Furthermore, after ligation of said target molecule to said adapter molecule, said resultant molecule is preferably circularized. Conveniently this is achieved by introducing the target molecule into a vector (or by attaching a portion of the target molecule to a support allowing free interaction after cleavage within the molecule, see Example 22) and using appropriate steps of cleavage and ligation after said adapter molecule has been introduced. Alternatively the chains of magnifying tags which are generated may be transferred or copied to a distant site on the target molecule without the need for effective circularization. An appropriate protocol for performing this is illustrated in Example 9.
Another convenient technique which avoids the need for excessive cycling involves the hybridization of smaller converted fragments, ie. nucleic acid molecules with attached magnifying tags. These fragments may themselves have been subjected to one or more conversion cycles and then may be linked by complementarity to unconverted sequences or information carried in the magnifying tags, e.g. nucleotide sequences of the tags (see Example 10).
To effect cycling of the reaction the control of particular enzymes used in the reaction is necessary. This may be achieved in different ways depending on the enzymes which have been used. Thus, methylation may be used to prevent binding to and/or cleavage at restriction sites. Ligation may be prevented or allowed by controlling the phosphorylation state of the terminal bases e.g. by appropriate use of kinases or phosphatases. Appropriately large volumes may also be used to avoid intermolecular ligations. Small volumes are preferably used during the restriction reactions to increase efficiency.
Preferably in each cycle of magnification (or sequencing as described herein), at least two bases are converted, preferably between 3 and 100, especially preferably from 4 to 20 bases per cycle. Conveniently, more than one magnifying tag is associated with one or more bases in each cycle. For example, in a preferred embodiment, a collection (e.g. a linear series or chain) of tags, each corresponding to one or more bases, collectively corresponding to a portion of said sequence, are introduced, e.g. multiple tags, e.g. more than 4 tags, corresponding to for example 4 to 12 contiguous bases. Conveniently this may be coupled with such tags themselves being directed to more than one base, e.g. unique tags for each pair of bases.
As will be noted in Example 1 in a preferred embodiment, a nuclease having the properties described above is employed to generate the overhang. In addition said vector additionally comprises a restriction enzyme site to produce a blunt end cleavage at one of the ends resulting from nuclease cleavage to produce the overhang. Alternatively, a restriction enzyme distinct to the enzyme used to create the initial overhang may be used which produces an overhang which has precise complementary to one terminal of all adapters employed in the reaction.
To perform the method of Example 3, conveniently nuclease sites which produce adjacent or overlapping regions of overhang are used. These sites are preferably located in the adapters which are employed. In each cycle two adapters are used which are conveniently allowed to ligate together by the use of complementary overhangs at the ends terminal to the regions binding to the single stranded portions of the target sequence. Thus in preferred aspects of the invention particularly to allow proof-reading adapters which are used comprise recognition sites for 2 or more nucleases with cleavage sites separate from their recognition sites, in which cleavage with said nucleases produces single stranded regions which are adjacent or overlapping. As used herein xe2x80x9coverlappingxe2x80x9d refers to sequences which have bases in common or which are complementary to such sequences, ie. on a corresponding strand. Thus, in order to achieve overlapping regions, use may be made of each strand of a double stranded target and overlapping, but complementary regions may be sequenced. Conveniently to achieve this effect more than one adapter is bound to the target molecule in each cycle. This method allows proof-reading if overlapping regions are sequenced as more than one magnifying tag corresponding to a particular base or collection of bases will become attached allowing the generation of a repeat signal for that base. It will be appreciated that in accordance with the invention one tag per base is not required and thus a tag for a pair of bases etc. may be repeated.
In performing the embodiment involving the use of Klenow fragment, said single stranded region which is created in step a) is one or more bases longer than a single stranded region of nucleic acid present on the adapter. Furthermore, an additional step is required after step b) in which the length of the single stranded region of the target molecule is shortened by polymerization extension reaction.
For performing techniques involving single stranded target molecules, cycling conveniently involves the generation of double stranded molecules, preferably by the use of the adapter as a primer in polymerase extension reactions.
The method in which recognition sites are completed to identify molecules having the terminal bases necessary to complete that site provides a slightly different technique to that described in general terms above, since the adapter binds to an overhang but carries tags which may not necessarily correspond to one or more bases of the single stranded region to which the adapter molecule binds. The single stranded region is made up of overhang created by cleavage of the restriction site which comprises some of the bases of the target sequence. However, depending on the cleavage site, those bases may or may not be in single stranded form, for example, the overhang may be entirely composed of non-target molecule bases. Instead the addition of the appropriate tag relies on the fact that adapters will only bind where the restriction site has been completed. Thus step b) includes reference to tags which correspond to one or more bases of said single stranded region or in proximity to said region, e.g. adjacent to said region. Furthermore in this method prior to step a), a piece of linker DNA comprising a part of a metabolic enzyme recognition site is attached to said target molecule, followed by use of said enzyme, e.g. nuclease to produce the single stranded form of step a).
As mentioned previously, sequencing may be performed on the basis of sorting. This method may be used independently of, or in combination with the above described magnification technique. For example, the sequencing protocol may be effected by sorting target nucleic acid molecules on the basis of four base pairs, and subsequently the adjacent base pairs may be converted to determine their sequence. For example, a sorting strategy can consist of creating overhangs with four bases in the target nucleic acid molecules as described previously. It is then distributed among 256 wells that are all covered by short DNA molecules, sorting adapters (these adapters do not necessarily carry magnifying tags). The sorting adapters are fixed to the well walls and have overhangs with four bases that can complement the overhangs that have been created on the target DNA. In addition, the sorting adapters may contain a binding site for an IIS enzyme or other appropriate nuclease. The binding site is located in such a way that the respective IIS enzyme can create an overhang with the base pairs that are located beside the first overhang that was created in the target DNA. In order to increase the surface area with sorting adapters, an alternative is to fix them to a solid support such as paramagnetic beads or similar.
The DNA molecules in well 1 have AAAA (SEQ ID NO:1) overhangs, while the DNA molecules in well 2 have AAAC (SEQ ID NO:8) overhangs, etc. The 256 wells thereby cover all permutations of overhangs on four bases. When the target DNA are added to the wells together with ligase, the DNA molecules with TTTT (SEQ ID NO:2) overhangs will attach themselves to well 1, the target DNA with TTTG (SEQ ID NO:9) overhangs to well 2, etc. After having washed off target DNA molecules that were not ligated to the sorting adapters, us enzyme is added so that the target DNA molecules are freed at the same time as a new overhang is created that represents the next four base pairs in the target sequence. This overhang can then be used as the starting point for a new round of sorting, or one may proceed with conversion/magnification.
Sorting strategies where DNA molecules are washed away involve a relatively large loss of DNA molecules. However, most sequencing protocols proposed in this patent application are based on the analysis of individual molecules, and this means that very few DNA molecules are required. Thus, even a loss of 99.9% or more seldom presents a problem.
Instead of using different wells, an alternative would be to use different positions on a xe2x80x9cmicroarrayxe2x80x9d. At address 1 it is only DNA molecules that end with TTTT (SEQ ID NO:2) that are fixed, at address 2 it is DNA molecules with TTTG (SEQ ID NO:9) ends that are fixed, etc. Other alternatives are to let DNA molecules with different ends attach/convert at different times, the use of gel sorting, etc.
For example, one may use a strategy where there are 256 different sorting adapters distributed among 256 squares on a xe2x80x9cmicroarrayxe2x80x9d. In square 1, there are sorting adapters with AAAA (SEQ ID NO:1) overhangs, in square 2, they have AAAC (SEQ ID NO:8) overhangs, etc. Thus, the target DNA molecules will be sorted so that those with TTTT (SEQ ID NO:2) overhangs are attached to square 1, GTTT (SEQ ID NO:10) overhangs to square 2, etc. By also fixing the other end of the DNA piece to the substrate, e.g. with biotin/streptavidin, one can then continue to the next conversion/magnification step without the DNA molecules leaving their position on the reading plate. Another strategy for preventing the DNA molecules from leaving their positions is to use a reading plate that is divided into 256 wells/spaces.
It must also be pointed out that sorting can, of course, be done with fewer or more permutations than 256. Sorting can also be performed in several rounds. For example, if one uses a xe2x80x9cmicroarrayxe2x80x9d with 65,536 different squares, it would be possible to identify eight bp by sorting through hybridization alone. This would be sufficient for many applications in order to perform a successful reconstruction. Sorting can therefore function as a sequencing method by itself, without having to use conversion or magnification.
Sorting can also be performed with non-ligase based strategies. In principle, one can use any method that is suitable for recognizing base pairs, including all the methods mentioned in connection with magnification.
It should also be pointed out that the specificity of a sorting method can be adjusted to most purposes by repeating the same sorting procedure one or several times. It may also be appropriate to use competing probes/overhangs in order to increase specificity.
Thus in a preferred aspect the present invention provides a method of sequencing a target molecule as described herein wherein said sequence is determined by assessing the complementarity of a portion of said molecule by a process comprising at least the steps of:
a) converting at least a portion of said target sequence to a form suitable for binding a complementary probe attached to a solid support or carrying a means for attaching to a solid support, preferably to single stranded form;
b) binding said complementary probe to at least a portion, preferably 4 to 12 bases in length, of said region suitable for binding a complementary probe, preferably said single stranded region created in step a);
c) optionally repeating steps a and b) wherein said complementary probe binds to an adjacent or overlapping region of said target molecule relative to the region to which the complementary probe of the previous cycle bound; and
d) determining the sequence of said target sequence by identifying the complementary probe(s) to which said target sequence bound.
As used herein xe2x80x9cprobexe2x80x9d refers to an appropriate nucleic acid molecule, e.g. an oligonucleotide or PNA molecule.
Additional steps may also be included, e.g. the complementary probe may act as a primer and in which case polymerase reactions may also be performed as necessary.
As mentioned above, this sorting technique is preferably preformed by the use of multiple complementary probes and preferably between 2 and 8, especially preferably 4 bases are identifiable per cycle although this information may only be collected at the completion of the sequencing reaction. Particularly preferably complementary probes with between 2 and 8, preferably 4 unique invariant bases are attached to different discrete sites on said solid support. In the second and subsequent cycles, target molecules which are bound to said probes are transferred to one or more further solid supports bearing complementary probes to sequence adjacent or overlapping regions of said target molecules. In order to achieve this step a) may be performed in an analogous manner to that described for the magnification process, ie. the probes may themselves contain a restriction site for a nuclease, e.g. a IIS enzyme, which cleaves outside its recognition site, such that an appropriate overhang is generated.
The above procedure may be coupled with the magnifying procedure such that sequencing may be performed by a combination of sorting and magnification, e.g. after step b), overhang may be generated as described about and adapters carrying appropriate magnifying tags may be used to bind to said overhangs. The sequence may then be determined by a combination of reading the magnifying tags and by identification of the probe to which the target molecules have bound. Thus in a preferred feature the present invention provides a method of sequencing as described herein wherein a portion of said sequence is determined by the magnification method described herein and an adjacent or overlapping portion is determined by the use of complementary probes as described herein.
In most cases the technique adopted for positioning of sequence portions will depend on how the target DNA is generated for sequencing, e.g. if it starts from a common point, or if it is generated by fragmentation which results in target molecules starting from different points.
Nucleic acid molecules for sequencing may be generated in different ways. By treating a small amount of DNA with DNAse, sonication, vortexing or similar techniques nucleic acid molecules may be fragmented into pieces. Such techniques are well known in the art, see for example http://dnal.chem.ou.edu/protocol_book/ protocol_partII.htm1 which describes protocols for random subclone generation. By adjusting the parameters of these techniques, it is possible to adjust the average size of the target DNA fragments (as a rule, the optimum is to have average sizes of a few hundred base pairs). The methods should also be relatively non-specific with respect to where they cut/break the DNA molecules so that statistically DNA pieces are obtained that are cut/broken in most places in the original sequence.
Studies show that the ends of the fragmented DNA molecules consist both of blunt ends and short overhangs of 1-2 bases. If desirable, the overhangs can be treated in such a way that they become blunt ends (Klenow filling-in, etc.).
Conveniently for performing preferred methods of the present invention, which relies on the production single stranded overhangs, nucleic acid molecules may be fragmented by procedures which produce such overhangs. As mentioned before, sonication, vortexing, and DNaseI create short overhangs. One can also use restriction enzymes that cleave non-specifically. Several studies have shown that IIS enzymes are particularly well suited to domain swapping tests where the DNA binding domain can be replaced. Therefore, new IIS enzymes can be created where the cutting domain is tied to a DNA binding domain that binds DNA non-specifically.
The overhangs generated by known IIS enzymes vary from xe2x88x925 to +6 bases. If overhangs of more than six bases are desired, it may be appropriate to use other systems/strategies. One possibility is to use nicking enzymes that produce nicks in dsDNA outside of their own binding site. Two binding sites for such a nicking enzyme, that have an internal distance of more than six base pairs and that are placed on either side of the double helix should produce an overhang of more than six base pairs. In addition to existing nicking enzymes, it may also be possible to create new nicking enzymes, for example by mutating IP and IIS restriction enzymes.
As an alternative to fragmentation, it is also possible to choose a strategy where fragment of the target sequence are produced with the aid of PCR or similar methods. For example, one can start with a known sequence on the target DNA and then use this area as a template for a primer in a polymerase extension. If a method is used that terminates the polymerase extension reaction at arbitrary sites, a DNA ladder is created, in which there are DNA molecules of many different lengths, but all having one end in common. Alternatively, short randomized primers can be used so that all possible combinations of fragments are produced from the target sequence. However, a limiting factor when using polymerase extension is the extension lengths of the various polymerases.
Magnification techniques may be coupled to sorting and conversion techniques described herein. For example adapters may be used as primers when binding to single stranded targets. Polymerase reactions may furthermore provide means of establishing the existence of complementarity between adapters and target sequences.
In one preferred embodiment of the invention, target molecules are fixed to solid supports. This may be achieved in a number of different ways. The target molecule may be designed to have attached to one or more moieties which allow binding of that molecule to a solid support, for example the ends (or several internal sites) may be provided with one partner of a binding pair, e.g. with biotin which can then be attached to a streptavidin-carrying solid support.
Target molecules may be engineered to carry such a binding moiety in a number of known ways. For example, a PCR reaction may be conducted to introduce the binding moiety, e.g. by using an appropriately labelled primer (see for example Example 17). Alternatively, the target nucleic acid may be ligated to a binding moiety, e.g. by cleaving the target nucleic acid molecule with a restriction enzyme and then ligating it to an adapter/linker whose end has been labeled with a binding moiety. Such as would particularly suitable if an IIS restriction enzyme is used that forms a non-palindromic overhang. Another alternative is to clone the target molecule into a vector which already carries a binding moiety, or that contains sequences that facilitate the introduction of such a moiety. Such methods could similarly be used to introduce position markers as described in more detail below.
Alternatively nucleic acid molecules may be attached to solid supports without the need to attach a binding moiety insofar as the nucleic acid molecule itself is one partner of the binding pair. Thus, for example short PNA molecules that are attached to a solid support may be used. PNA molecules have the ability to hybridize and bind to double strand DNA and the undissolved nucleic acid material can therefore be attached to a solid support with this strategy. Similarly, oligonucleotide probes may be-used to bind complementary sequences to a solid support. Such a technique may also be used to begin sequencing by binding particular nucleic acid molecules to particular locations on a solid support as described below.
Appropriate solid supports suitable as immobilizing moieties for attaching the target molecules are well known in the art and widely described in the literature and generally speaking, the solid support may be any of the well-known supports or matrices which are currently widely used or proposed for immobilization, separation etc. in chemical or biochemical procedures. Thus for example, the immobilizing moieties may take the form of beads, particles, sheets, gels, filters, membranes, microfibre strips, tubes or plates, fibres or capillaries, made for example of a polymeric material e.g. agarose, cellulose, alginate, teflon, latex or polystyrene. Particulate materials, e.g. beads, are generally preferred. Conveniently, the immobilizing moiety may comprise magnetic particles, such as superparamagnetic particles. In a further preferred embodiment, plates or sheets are used to allow fixation of molecules in linear arrangement. The plates may also comprise walls perpendicular to the plate on which molecules may be attached.
Attachment to the solid support may be performed directly or indirectly and the technique which is used will depend on whether the molecule to be attached is a probe for identifying the target molecules or the target molecules themselves. For attaching the target molecules, conveniently attachment may be performed indirectly by the use of an attachment moiety carried on the nucleic acid molecules and/or solid support. Thus for example, a pair of affinity binding partners may be used, such as avidin, streptavidin or biotin, DNA or DNA binding protein (e.g. either the lac I repressor protein or the lac operator sequence to which it binds), antibodies (which may be mono- or polyclonal), antibody fragments or the epitopes or haptens of antibodies. In these cases, one partner of the binding pair is attached to (or is inherently part of) the solid support and the other partner is attached to (or is inherently part of) the nucleic acid molecules. Other techniques of direct attachment may be used such as for example if a filter is used, attachment may be performed by UV-induced crosslinking. When attaching DNA fragments, the natural propensity of DNA to adhere to glass may also be used.
Attachment of appropriate functional groups to the solid support may be performed by methods well known in the art, which include for example, attachment through hydroxyl, carboxyl, aldehyde or amino groups which may be provided by treating the solid support to provide suitable surface coatings. Attachment of appropriate functional groups to the nucleic acid molecules of the invention may be performed by ligation or introduced during synthesis or amplification, for example using primers carrying an appropriate moiety, such as biotin or a particular sequence for capture.
As described herein target molecules are conveniently attached to complementary probes which are attached to the solid support.
In techniques using multiple but discrete complementary probes the solid supports to which these different probes are attached are conveniently physically associated although the signals generated by attachment of a target molecule to each probe must be separately determinable. Thus for example, plates with multiple wells may be used as the solid support with different probes in the different wells, or regions of a solid support may comprise the different addresses, for example the different probes may be bound to a filter at discrete sites.
Attachment to a solid support may be performed before or after nucleic acid molecule fragments have been produced. For example target nucleic acid molecules carrying binding moieties may be attached to a solid support and thereafter treated with DNaseI or similar. Alternatively cleavage may be effected and then the fragments may be attached to the support.
In many contexts, the object is to sequence one or several sequences that are present inside or together with other sequences. For example, only 5-10% of human genome sequences are assumed to be of direct biological importance. For mass screenings of human genomes, it would therefore be useful to be able to avoid sequencing areas that are of minor biological importance.
Thus one strategy which may be used is to fix polynucleotides that complement the target sequences that are to be isolated to a solid support (the inside of a well, mono-dispersed spheres, microarrays, etc.). By hybridizing the polynucleotides in the sequence pool with polymers on the solid support, undesirable polynucleotides can be washed away before proceeding to the sequencing stage. If desired, specificity can be increased by performing several cycles of hybridization and washing. Even if it may be advantageous for individual applications, there is no dependence on whether the complementary polynucleotides are fixed in a regular pattern. Similar strategies based on ligation, PNA hybridization, etc. are also possible.
For example, to isolate specific mRNA/cDNA molecules, complementary cDNA/mRNA molecules can be fixed to paramagnetic spheres or the like. The spheres can then be mixed in a tube together with the solution that contains the target sequences. When the mRNA/cDNA molecules have been hybridized with the mRNA/cDNA that are fixed to the spheres, undesirable molecules can be washed away at the same time as the spheres are kept in the tube with a magnet or similar. The desired target molecules can then be released by increasing the temperature, changing the pH, or by using another method that dissolves the hybridized molecules.
A similar strategy that can be used for sequencing protocols done on a reading plate is to fix specific target sequences to determined addresses. For example, single strand target DNA can be hybridized to primers that are fixed to different addresses. If desired, the primers can then be used as templates for a polymer extension. By adjusting the primers to the target sequence, it can be addressed as desired.
A corresponding strategy can be to fix PNA molecules to the different addresses. PNA molecules are known to have the ability to recognize specific sequences in dsDNA and such a strategy can therefore be used to address dsDNA by using PNA molecules that recognize the sequences that one wants to fix.
As mentioned above, molecules which are sequenced which may be divided into two categories. Those which have one common end and one arbitrary end and those which have two arbitrary ends. Positional information may be obtained from these different types of molecules in different ways.
If all the target molecules have a common end, the length of each target molecule will be proportional to the distance between the common end and the other arbitrary end. Similarly, sequence information that is attributed to a particular portion of that target molecule may be positioned by calculating the distance from the common end to the site of the sequence information. Conveniently where that sequence information relates to the end of the target molecule, its position may be determined from the length/size of the entire molecule.
If nucleic acid fragments do not start from a common end, positional information may be obtained in different ways. One alternative is to create or identify characteristic fingerprints that vary from sequence to sequence. Thus, the position of a sequence piece can be derived by registering what fingerprint it is tied to, and possibly where in the fingerprint it is located. Very many techniques can be considered for use for creating characteristic patterns. The cleavage pattern of restriction enzymes in a DNA sequence can be registered, e.g. with the aid of xe2x80x9coptical mappingxe2x80x9d or similar methods.
One disadvantage of known xe2x80x9coptical mappingxe2x80x9d methods is that the cutting sites for the restriction enzymes that are used are not always cut. Likewise, incorrect cleavage can occur and there may be some uncertainty associated with the length measurements of the DNA fragments. Therefore, it is necessary to produce an average picture of each map piece based on an analysis of many identical DNA molecules. The problem is that it can be difficult to know what DNA molecules are identical.
Another problem with current methods of optical mapping is that treatment with restriction enzymes and the like must take place after the DNA molecules have been straightened out in order to be able to observe the internal placement of the DNA fragments. This reduces the availability of the DNA molecules for such things as enzymatic preparation. The present invention which provides end terminal sequencing in addition to positional placement allows such problems to be overcome. See for example the technique described in Example 23.
One can also use fluorescing probes/tags that create characteristic patterns. This is the principle behind the so-called xe2x80x9cDIRVISHxe2x80x9d technique. A similar strategy is to use atomic force microscopy (AFM), micro-/nanopores, or other methods for registering the size and location of proteins that are bound in characteristic patterns, etc.
One can also use cellular adapters as discussed previously. For example, if one transforms/transfects magnified target DNA into cells, one can take advantage of the fact that the transcription frequency of a reporter gene varies with the distance to cis-regulatory elements. If there is an enhancer at one end and one or more magnifying tags at the other consisting of reporter genes, the relative quantity of reporter proteins can be used to calculate the position value.
It is also possible to label or incorporate the target sequences with elements that are used to derive the position value. Such strategies can be advantageous, e.g. if it is difficult to distinguish between the fingerprints of two very similar sequences. For example, if one wishes to sequence sister chromosomes, one can integrate a large number of insertion elements (transposons or the like) that are arbitrarily integrated. If one then amplifies the chromosomes and use the insertion elements as position markers, there will be one or several characteristic patterns for each sister chromosome.
An alternative strategy that may be used which can introduce both a positional marker and allows identification of a sequence at that site involves the use of adapters as primers for a PCR reaction. The result of each PCR reaction will be two adapters that are connected, where the distance between the two adapters corresponds to the distance of the adapter sequences on the target DNA and simultaneously provides positional information.
The target molecule""s sequence may provide the necessary means for producing a position marker without modification. For example, if some sequence information is known, a probe may be used to hybridize to that sequence which then provides a position marker. Alternatively, appropriate position markers may be placed into a target molecule, e.g. different position markers may be placed at regular intervals in a genome. To allow discrimination between the different position markers different signals are provided by those markers, e.g. they have different sequences or lengths which may be probed. Example 21 describes one method in which position markers are used.
Thus viewed in a preferred aspect the present invention provides a method of sequencing (completely or partially) a nucleic acid molecule comprising at least the steps of:
a) determining the sequence of a portion of said nucleic acid molecule;
b) determining the position of said portion within said nucleic acid molecule by reference to a positional indicator, preferably a position marker; and
c) combining the information obtained in steps a) and b) to obtain the sequence of said molecule.
As mentioned previously, multiple sequences and their positions are preferably determined.
As used herein the positional indicator may as mentioned previously be the size of the molecule, the intensity of a generated signal or the distance to a positional marker, anchor or fingerprint.
A number of different techniques for performing these methods will now be described to illustrate the invention.
Every method for determining the size of polymers can be used, in principle. The length of the sequence pieces that are identified must, however, be adjusted to the precision of the size determination: the lower the precision, the longer the sequence pieces must be.
A number of methods for size sorting exist in the field; gel sorting, micro-capillary sorting, measuring of the lengths of polymers that are stretched out on a reading plate, measuring of the fluorescence intensity (or other) of polymers that are non-specifically tagged with the aid of a flow cytometer, fluorescence microscope, etc.), mass spectrometry, the time a polymer uses to block a micro or nanopore, etc. Such procedures may be conducted before or after the signal is read to determine the sequence, e.g. when gel electrophoresis is used, reading may be performed on samples separated on a gel or eluted from the gel.
The length of a nucleic acid molecule may also be determined based on the principle that the chance that a DNA molecule will be cleaved (e.g. by DNaseI, sonication etc) is proportional to the length of the DNA molecule. For example, a DNA molecule with 200 base pairs will be cut twice as often as one with 100 base pairs in a solution with a limiting amount of DNaseI. This could be achieved for example by end labelling different molecules, subjecting them to cleavage and then monitoring the amount of single and double labelled molecules relative to standards of known length similarly labelled.
In order to determine the length of a DNA molecule or the distance to a fixed point, the DNA molecule may conveniently be extended or stretched out. One method of stretching the DNA molecules is to mix them with a large surplus of small glass beads (they bind DNA molecules naturally) so that they bind the DNA molecules in a 1:1 ratio. The DNA molecules will have less resistance than the glass beads in the liquid flow so that they tend to move away from each other until the DNA molecule is stretched out. If the liquid flow is strong or the glass beads are large so that the difference in resistance between the DNA molecules and the glass beads is great, the DNA molecule may tear. However, this problem can be avoided by lowering the flow speed or by using smaller glass beads. The method becomes particularly efficient if the DNA molecules are arranged in a regular manner so that the amount of sequence information is increased per unit area. One way of doing this is to label the DNA molecules with biotin and then fix them to a plate with a regular streptavidin pattern. Alternatively fixing may be achieved using a laser beam, so-called laser trapping.
Instead of using a liquid flow to straighten out the DNA molecules, one can use a positive charge that pulls the negatively charged DNA molecules in one direction. Reading efficiency is likely to increase by using this strategy. The easiest method, in principle, is to place a positive or negative spot charge in front of the reading plate. According to Coulomb""s law, the force of the charge on the DNA molecules is inversely proportional to the distance. The DNA molecules closest to the charge will then be stretched with a greater force than those that are further below. In order for all DNA molecules to be equally affected at the moment of reading, it will therefore be necessary to move the spot charge in step with the reading unit. The spot charge can also be placed far below the reading plate so that the force difference on the plate is reduced. As an alternative, it is possible to arrange the charge in an arc so that the force vectors are equally large in a straight line at the centre of the arc. Then the charge needs only to be moved when the reading unit is moved sideways.
Alternatively, to reduce the force on the molecule""s anchor a different technique may be used. Two electrically charged plates may be placed under the reading plate on which the target molecules are to be stretched out. The top plate has a weak negative charge while the bottom plate has a relatively strong positive charge. If a negatively charged particle (e.g. DNA) is placed right above the negative plate, the repulsion forces from it will be greater than the attraction forces from the positive plate. The particle will then be forced upwards. However, moving away from the plate conditions will be reversed. The attraction force of the positive plate is greater than the repulsion force of the negative plate. By adjusting the charges of the plates, equilibrium will occur between repulsion and attraction forces at a given height above the reading plate. The target molecules will be pushed into this plane of equilibrium. In this method the net force on the DNA molecules is equal to zero as long as they remain in the plane of equilibrium. This reduces the chance of rupture.
In addition to the two charged plates a positive charge to the left of the reading plate may also be used. This will produce a net force in this direction. The same can be achieved by tilting the two charged plates in relation to each other and in relation to the reading plate.
If target molecules are to be moved while stretched through a flow cytometer or similar device, a negatively charged tube may be used. By using such a technique, target molecules will be pushed in towards the middle of the tube where the repulsion forces are the weakest.
A further alternative stretching technique is provided by mechanical stretching. In this method for example, two adjacent plates may be used in which oligonucleotides complementary to either end of the target molecules are attached. Once target molecules have been hybridized to these probes, the plates may be separated until the molecules are stretched between them.
The signal generated in the above described methods may be read in a number of different ways, depending on the signal which is generated and how positional information is to be obtained. For example to locate fluorescent DNA probes attached to a target DNA, the DNA may be stretched as described above. For example a method developed by Weier et al. (Hum. Mol. Gen., 1995, Vol. 4(10), p1903-1910) known as molecular combing may be used. In this method a solution with target DNA was placed on a flat glass surface prepared so that the DNA molecules attached themselves with one end to the glass plate. The DNA molecules were then straightened out by using a liquid flow. With the aid of a fluorescence microscope they could then observe the relative positions of the probes which were attached to the stretched DNA molecules.
In the present invention, by for example using four probes labelled with different fluorophores and magnifying tags which are unique stretches of DNA, the probes may be directed to those tags such that they hybridize to the four magnifying tags that represent A, C, G, and T, ie. using the DIRVISH techniques described previously. The sequence order may then be read directly with a fluorescence microscope. As mentioned previously, more or fewer probes may be used depending on how the magnifying tags are constructed, e.g. a single probe may be used in which the manner in which it binds to each magnifying tag produces a unique signal, e.g. the development of a binary code. Alternatively, more than 4 probes may be used where the magnifying tag corresponds to 2 or more bases. By developing software that causes the microscope to scan the glass plate while at the same time automatically analyzing the sequence order, it will be possible to read base pairs very rapidly.
As a further alternative, for fast reading a flow cytometer may be used to read the fluorescent probes. A prerequisite for this is that the DNA molecules pass the reading unit of a flow cytometer in a stretched form so that the magnifying tags that represent A, C, G, and T will pass in order. This may be performed by using the techniques described above. Alternatively for this particular embodiment, an electric or magnetic field may be used instead of liquid flow to pull the particles past the fluorescence detector. This can be achieved by utilizing the fact that the glass beads have a positive charge while the DNA molecules are negatively charged, or to use superparamagnetic beads instead of glass. The beads would then pull the DNA molecules behind them like long threads.
A critical parameter in this strategy is the lower fluorescence detection limits of the flow cytometer. Several groups have managed to detect individual fluorophore molecules by reducing flow speed. However, to use conventional flow cytometers with analysis speeds of 20-30,000 particles per second, longer probes must be used so that many fluorophores can be fixed to each probe.
The fastest flow cytometers currently have the capacity to analyze about 200,000 fluorescent particles per second, but these flow cytometers are not commercially available. In addition, it is not certain what the high-speed tolerance of DNA molecules is in stretched form before they break. However, it is realistic to assume that the DNA molecules will tolerate speeds that will allow extremely rapid reading.
A further alternative is to fix the DNA molecules in a regular fashion on a solid support, e.g. a streptavidin-covered plate. The sequence (e.g. the signals generated by a series of magnifying tags) is read by having small detectors inserted in the reading plate. These detectors are deactivated or activated by reporter molecules, e.g. on the magnifying tags, fixed to the fragments, e.g. by breaking or establishing electrical circuits by binding to sensors on a solid support. For example, strong bonds may be formed between the reporter molecules and modules on the reading plate. In the latter case the modules may be shaped in such a way that they can be torn loose from the reading plate if the DNA molecules are torn away. When the modules come loose, they break the current circuit in a way that registers what modules have been removed from the reading plate. In order to increase the chance of a successful binding, several reporter molecules may be fixed in the same position on the fragment. One could either use four different reporter molecules for each of the bases A, C, G, and T, or use the same reporter molecule positioned on four different places on the fragments. With multiplex, parallel computer inputs and other modern electronics, it is believed to be possible to register several million signals per second allowing rapid sequencing.
In a preferred embodiment these methods are appropriately used in conjunction with the magnifying techniques described herein, ie. a portion of said target nucleic acid molecule is determined by the presence of one or more, preferably a chain of magnifying tags. These methods may however also be used when no magnification is performed. Instead of magnifying the DNA molecules, one could incorporate different attachments to the bases that the sensors can register.
Once the signal information has been accumulated, a computer program is used to assemble the sequence pieces into the final sequence. The probability that errors will occur in this step depends primarily on five parameters: the length of the DNA molecule that is to be sequenced, how randomized the base pair composition of the DNA sequence is, the length of the DNA pieces that are to be read, the number of DNA pieces that are being read, and the error rate in the sequencing reactions.
The inventor has already created a computer program to analyze the importance of the above-mentioned parameters. Based on human genome DNA that has already been sequenced, the analyses show that with a DNA piece length of 30 fragments, reading of 6xc3x97108 DNA pieces, and an error rate in the sequencing reaction of 10% (considering spot mutations), a human genome could be read in a single sequence reaction and with very few spot mutations/deletions. However, one exception is very non-randomized areas (satellite DNA and other repetitive areas) where the DNA piece lengths must be increased. The biological information in these areas, however, is of subordinate importance compared to coding sequences and cis-regulatory elements.
Data analyses also show that even a very high error rate in the sequencing reaction is compensated for when the DNA pieces are read many times. For example, by reading ten times as many base pairs as the length of the sequence, most deletions and spot mutations will be eliminated even with a high error rate in the sequencing reaction.
Depending on the technique which is used for sequencing it is in certain circumstances possible to perform sequencing on a heterogeneous sample, e.g. to perform parallel sequencing. Procedures which allow this form preferred aspects of the invention. Such techniques requires that signals from different target molecules may be discriminated. This may be achieved in a number of ways, e.g. by restriction to particular locations, inclusion, or identification, of markers which identify particular target molecules etc. For example, solid supports which complement a region of a target nucleic acid molecule may be used to isolate and retain a particular molecule. This may be performed with knowledge of at least a part of a sequence, ie. to bind particular molecules to a particular site, or without such knowledge using essentially random binding probes on which different molecules will bind and which may then be sequenced in parallel, by making use of one or more techniques to relate the sequence to that molecule, e.g. by address or positional marker. The techniques described herein are particularly advantageous since they allow individual molecules to be sequenced thus further aiding the facilitation of parallel sequencing reactions.
A number of the techniques described herein may be used for sequencing only a part of a target molecule or for fingerprinting, profile analyses or mapping, ie. identifying discrete and distinctive portions of a molecule, e.g. for analysis of RNA expression (which may first be converted to cDNA for analysis). For example, as described in Example 23, a target sample may be digested with a restriction enzyme which produces a particular overhang to which a magnifying tag (preferably a chain of magnifying tags) may be attached. In addition to carrying sequence related information, such tags may additionally carry information relating to the enzyme which resulted in cleavage, ie. as a marker of fragments resulting from that cleavage. More than one restriction enzymes may be used simultaneously if these produce different length overhangs to which different adapters may be bound. Alternatively different restriction enzymes may be used in consecutive cycles.
The resultant fragments may then be aligned for example by virtue of the amplifying tags attached to complementary overhangs, e.g. by the use of tags which reflect this complementarity, e.g. wherein the tags themselves are made up of nucleotide bases. From this a restriction map may be built up as described in Example 23. Thus in a further aspect the present invention provides a method of producing a map of a target sequence comprising obtaining sequence information on discrete portions of said sequence as described herein in addition to positional information on said portions as described herein.
In a preferred feature said map is produced by obtaining sequence information on discrete portions of said sequence wherein said portions comprise all or part of the cleavage sites of one or more nucleases and/or all or part of the restriction sites of said nucleases and the positions of said sequences are determined by comparison of the sequences at the terminal ends of fragments of said target nucleic acid molecule after digestion with said nucleases.
Preferably sequence information is obtained by cleavage of said target molecule by one or more nucleases as described herein, preferably to produce complementary single stranded regions, and binding of an adapter molecule to a region of said target molecule (preferably at, or adjacent to the cleavage site) wherein said adapter molecule carries one or more magnifying tags as described herein wherein said tag comprises a signalling moiety which corresponds to one or more bases of said region to which said adapter molecule binds and additionally comprises a further signalling moiety which corresponds to the nuclease used for cleavage. In instances in which sufficient nucleases are employed, this method may be used as a method of sequencing.
For example, a bacterium may be identified in the following manner. Bacteria may be lysed and the DNA isolated. The molecules may then be cut with a class II restriction endonucleases or other such nucleases as described herein. The DNA molecules may then be bound to adapters to identify the overhangs. The DNA molecules may then be fixed to a reading plate and stretched. By scanning the reading plate with a fluorescence scanner information or characteristic pattern may be obtained on restriction lengths and sequence information derived from the ends of those molecules. Techniques in which such magnification is involved allows the discrimination of molecules of the same size by virtue of their end sequences. Thus in a further feature of the invention, the present invention provides a method of obtaining a fingerprint of a target DNA molecule comprising the use of one or more of the sequencing techniques described herein in addition to obtaining positional information as described herein.
In preferred features of the invention, positional information is obtained by reference to a characteristic restriction map of said target molecule. Further preferred features include the use of restriction mapping to identify one or more magnifying tags and the use of tags which may effectively be read using flow cytometers or nano/micro pore analysis.
Using the principles and protocols that are introduced in this patent application error rates can be reduced by using proofreading techniques both in the sequencing/sorting reactions and when reading the signals.
If sorting is used, it is possible to sort the same piece of sequence several times. For example, all target DNA that begin with AAAA (SEQ ID NO:1) are sorted into well 1. Then the same procedure is repeated where incorrectly sorted DNA molecules that do not end with AAAA (SEQ ID NO:1) are washed away. The procedure can in principle be repeated until the desired error percentage is obtained.
If magnification/conversion is used, it is possible to convert the same piece of sequence on a target molecule several times so that a repetitive chain of magnifying tags (signal chain) is obtained. Most error conversions can then be discovered when the repeated conversion products are not alike. The portion of a target molecule that is to be used to derive position information can also be copied in the same manner.
In addition, each sequence piece may be read many times because the number of target molecules that are analyzed can be very great.
Kits for performing the sequencing or magnification methods described herein form a preferred aspect of the invention. Thus viewed from a further aspect the present invention provides a kit for magnifying one or more bases of a target nucleic acid molecule comprising at least one or more adapters as described hereinbefore, optionally attached to one or more solid supports, preferably comprising one or magnifying tags themselves comprising a signalling means.
Optionally the kit may contain other appropriate components selected from the list including restriction enzymes for use in the reaction, vectors into which the target molecules may be ligated, ligases, enzymes necessary for inactivation and activation of restriction or ligation sites, primers for amplification and/or appropriate enzymes, buffers and solutions. Kits for carrying out other aspects of the sequencing reactions described herein are also included within the scope of the invention. Thus for example kits for performing the sorting reaction may comprise at least a solid support carrying one or more complementary probes, preferably a series of discrete probes mismatched to each other probe at a different address on the solid support, or on a separate solid support, by one or more bases. Appropriate labelling means may also be included in such kits.
The use of such kits for magnifying target nucleic acid molecules or for sequencing form further aspects of the invention.